1. Field of the Invention
This invention relates to the field of biomimetic or lipid-based self-assembling molecular materials and to color-changing biosensing microdevices.
2. Description of the Related Art
Since the early 1980s, considerable attention has been directed towards the fabrication of amphiphilic lipid based self-assembling materials as membrane mimetics for a wide range of applications (Fendler, J. H. Biomimetic Membranes: Wiley, New York, 1982; Fendler, J. H. Science 1984, 223, 888–894; Ringsdorf, H., et al., Angew. Chem. Int. Ed. Engl. 1988, 27, 113–158). Today, these materials are playing important roles in the construction of biosensors (Charych, D. et al., Science, 1993, 261, 585–588; Charych, D. et al., Chem. Biol. 1996, 3, 113–120; Song, X. et al., J. Am. Chem. Soc. 1998, 120, 4813–4814 and 11314–11315), controlled release systems or drug/gene delivery vehicles, synthetic supramolecular immunogens and many other nanomachines. Despite the continuing emergence of new applications, however, fundamentals, such as the relationship between the microscopic morphology of the self-assembled systems and the chemical structure and conformation of their constituent lipids, remain to be elucidated. On the practical side, it continues to be challenging to rationally design well-defined functional materials and quickly access highly ordered assemblies under mild conditions.
One application of self-assembling materials is in the field of biosensors. Previous calorimetric sensors were constructed using polydiacetylene lipids incorporating cell surface receptors (e.g. sialo-lipid or GM1 ganglioside). These were assembled into films for the detection of influenza virus (Charych et al., Science 1993, 261, 585–8) and a number of bacterial toxins (Charych et al., Chem. Biol. 1996, 3, 113–20). See U.S. Pat. Nos. 6,001,556; 6,022,748; 6,080,423. These sensors integrated molecular recognition and signal transduction into one supramolecular assembly. The conjugated polymer backbone provided the signal transduction pathway and responded to binding events by a straightforward color change. The detection feature of these colorimetric sensors allows for unaided visual on-site detection of biological hazards and offers great potential for a variety of household medical or diagnostic applications. Thus biosensors can also aid in the detection of the presence or absence of a virus, bacteria, disease indicators, compounds, etc.
More recently, other cellular components have been coupled to polydiacetylene Langmuir films and liposomes, such as nucleic acids (U.S. Pat. No. 6,306,598), proteins (co-pending U.S. patent application Ser. No. 09/023,898) and amino acids (See Song et al., Langmuir 2000, 120, 4873–4874) to create biosensors that allow the detection of biological ligands and analytes.
There are, however, limitations to the use of these amphiphilic lipid-based thin film or vesicle sensors. For example, the fabrication of the thin film sensor requires the Langmuir-Blodgett technique, and the detection sensitivities obtained are yet to be further improved. Typically, layered lipid materials must be painstakingly coaxed to assemble, sometimes under conditions much different than those found in nature, and they have limited stability. Thus, a more stable (less fluid) lipid assembly could increase the signal transduction efficiency of biosensors.
There is also a long standing interest of applying self-assembling amphiphilic lipids to coat traditional metallic bone implants to enhance directed biomineral growth and tissue integration at the tissue-implant interface (Berman et al., Science 1995, 269, 515–518). However, such biomimetic approach can only be limited to surface modification. A self-assembling bulk polymer that is capable of inducing biomerialization as well as functioning as structural, rather than surface, scaffold of bone implants is not yet available.
A bolaamphiphilic lipid consists of a hydrocarbon chain as the hydrophobic core, and a hydrophilic polar headgroup on each end of the hydrocarbon chain. It is also referred as a bisfunctional lipid in this invention. An amphiphilic lipid, on the other hand, consists of one hydrocarbon chain as the hydrophobic tail and a hydrophilic headgroup on one end of the hydrocarbon chain. It is also referred to a monofunctional lipid.
Some bacterial lipids are transmembranic, coupled tail-to-tail, with functional headgroups on both the inside and outside surfaces of the cell's membrane. Such a two-headed structure resembles an Argentine gaucho's bola, a rope with a weight at each end—thus the adjective, “bolamphiphilic”.
Bolaamphiphilic lipids, or bisfunctional lipids, as employed in the present invention, complement very well many of the limitations of monofunctional lipids and provide improved properties. Some of the improved properties noted are that bolaamphiphiles are capable of forming stable structures such as vesicles, and can maintain fluid phases at fairly large surface areas per molecule (Meglio et al., Langmuir 2000, 16, 128–133; Escamilla et al., Angew. Chem. Int. Ed. Engl. 1994, 33, No. 19:1937–1940; Bader et al., Faraday Discuss. Chem. Soc., 1986, 81, 329–337). Bolaamphiphiles tend to form well-defined microstructures under mild conditions and have high biological relevance as mimics of natural transmembrane lipids' such as those isolated from the thermophilic anaerobic eubacterium, Thermoanaerobacter ethanolicus 39E (Jung et al., J. Lipid Res. 1994, 35, 1057–1065). In nature, membrane-spanning bipolar lipids provide extraordinary stability to archaebacteria, a class of microorganisms that resist extreme environmental conditions such as low pH, high temperature, and high salt (Langworthy, T. A., Curr. Topics in Membr. Transp. 1982, 17, 45–77). Furthermore, the bacterial bilayer in several species of thermophilic bacteria has been found to undergo structural reorganization in response to these extreme conditions (Lee et al., J. Am. Chem. Soc. 1998, 120, 5855–5863). Finally, when structurally different functional groups are installed at the two ends of bolaamphiphiles, for the fabrication of materials with asymmetric interfacial properties properties can be achieved. For instance, when one end of a bolaamphiphile is functionalized with a thio group while the other end modified with a sialogroup, they can be immobilized onto gold surface (via Au—S bond) for the electrochemical detection for influenza virus (via the sialo terminus).
Various studies have suggested that the physical nature of the lipid matrix plays a dominant role in the vesicular budding and fission process (Ringsdorf et al., Angew. Chem. Internat. Ed. Engl. 1988, 27, 113–158; Dobereiner et al., Biophys. J. 1993, 65, 1396–1403). It is known that both general thermodynamic constraints and the geometry of each amphiphilic molecule present in a lipid matrix are crucial factors in determining the final shape and morphology of the aggregates formed (Israelachvili et al., J Chem. Soc.—Faraday Trans. II 1976, 72, 1525–1568). Specifically, chirality and the appropriate geometry of constituent lipids are crucial determinants for the chiral packing of self-assembling materials, which has been considered by many as the driving force for tubular and helical microstructure formations (Thomas et al., Phys. Rev. E 1999, 59, 3040–3047; Schnur, Science 1993, 262, 1669–1676; Eckhardt et al., Nature 1993, 362, 614–616; Viswanathan et al., Nature 1994, 368, 440–443; Selinger et al., Phys. Rev. E 1996, 53, 3804–3818; Oda et al., Nature 1999, 399, 566–9). A number of polystyrene block copolymers have been studied by TEM for their rod-to-vesicle and vesicle-to-rod transitions induced by solvents and dilution (Yu et al., Langmuir 1999, 15, 7157–7167; Chen et al., J. Phys. Chem. B 1999, 103, 9488–9497). Shear flow-induced, surfactant-based vesicle-to-wormlike micelle and micelle-to-vesicle transitions have been studied by using small angle neutron scattering and TEM (Zheng et al., J. Phys. Chem. B 2000, 104, 5263–5271; Oberdisse et al., J. Phys. Chem. B 1998, 102, 1102–1108; Mendes et al., J. Phys. Chem. B 1997, 101, 2256–2258; Escalante et al., Langmuir 2000, 16, 8653–8663). Lipid doping effects on microstructure transitions, however, are relatively less explored (Schröder and Schürholz, Eur. Biophys J. 1996, 25, 67–73), especially for polymerizable bolaamphiphilic self-assembling systems.
One fundamental consideration in designing biosensors is the requirement of balance between the rigidity and flexibility of the sensor scaffold. Often times this balance is reflected in the microscopic morphology and the extent of polymerization (which is directly influenced by molecular packing) of the sensor material.
The potential of polymerizable self-assembling bolaamphiphilic lipids to form bulk polymeric material with ordered molecular arrangements at the polymer surface makes them ideal structural templates for applications such as, tissue engineering, especially for the engineering of organic-inorganic composites such as bone.